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EP2180880A2 - Encapsulation d'agents thérapeutiques dans des micelles - Google Patents

Encapsulation d'agents thérapeutiques dans des micelles

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Publication number
EP2180880A2
EP2180880A2 EP08780062A EP08780062A EP2180880A2 EP 2180880 A2 EP2180880 A2 EP 2180880A2 EP 08780062 A EP08780062 A EP 08780062A EP 08780062 A EP08780062 A EP 08780062A EP 2180880 A2 EP2180880 A2 EP 2180880A2
Authority
EP
European Patent Office
Prior art keywords
aag
micelles
poly
composition
peg
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP08780062A
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German (de)
English (en)
Inventor
Glen S. Kwon
M. Laird Forrest
Neal M. Davies
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Individual
Original Assignee
Individual
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Filing date
Publication date
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Publication of EP2180880A2 publication Critical patent/EP2180880A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Heat Shock Protein 90 is an important target for cancer therapy due to its key role in regulating proteins that are involved in tumor cell proliferation. It was discovered that geldanamycin, a benzoquinone ansamycin antibiotic, can strongly bind to the ATP/ ADP binding pocket of Hsp90, interfering with the survival and growth of a diverse family of tumors. Geldanamycin is a promising new anticancer agent, but its clinical development has been hampered by severe hepatotoxicity and poor solubility.
  • 17-allylamino-17-demethoxygeldanamycin (17-AAG; tanespimycin) and 17-dimethylamino-ethylamino-l 7-demethoxygeldanamycin (17-DMAG)
  • 17-DMAG 17-dimethylamino-ethylamino-l 7-demethoxygeldanamycin
  • 17-DMAG is significantly less than that of 17-AAG (8 mg/m /day and 100-200 mg/m /day in dogs, respectively).
  • the major obstacle for delivery of 17-AAG is its limited aqueous solubility (about 0.1 mg/mL), which has resulted in the use of complicated formulations with Cremophor ® EL (CrEL), DMSO, and/or ethanol before parenteral administration. This is undesirable from a patient tolerability standpoint because CrEL is known to induce hypersensitivity reactions and anaphylaxis, and requires patient treatment with antihistamines and steroids before administration.
  • the invention provides active agents, such as 17-AAG (17-allylamino-17- demethoxygeldanamycin), encapsulated by safe poly(ethylene g ⁇ yco ⁇ )-block- poly(lactic acid) (“PEG-PLA”) micelles.
  • PEG-PLA safe poly(ethylene g ⁇ yco ⁇ )-block- poly(lactic acid)
  • the compositions of the invention therefore provide effective solubilization of the active agents.
  • a significant advantage of PEG-PLA as a carrier is that it is less toxic than Cremophor ® EL or DMSO, which are used in currently known compositions.
  • PEG- PLA micelles are easier to handle than DMSO and they do not possess a foul odor, which is a problem with 17-AAG formulations currently in clinical trials.
  • Micelle encapsulation can also reduce the occurrence of side effects (e.g., hepatotoxicity, neutropenia, neuropathy, and the like) of certain agents by maintaining the agents within the micelles until they are delivered to
  • the PEG-PLA micelles can be used to encapsulate 17-AAG and/or a second active agent to provide a pharmaceutical composition.
  • the second active agent can be a chemotherapeutic agent, such as paclitaxel, docetaxel, teniposide, or etoposide.
  • the composition can be formulated to be suitable for various forms of internal administration, such as intravenous (IV) injection or infusion.
  • IV intravenous
  • Such formulations can include 17-AAG-containing micelles that have a 17-AAG concentration of up to about 20 mg/mL.
  • the formulations can include a suitable aqueous carrier, such as a saline or dextrose solution.
  • the invention also provides a method of administering 17-AAG to a patient in need thereof, comprising administering an effective amount of a composition that includes 17-AAG encapsulated within micelles.
  • the micelles can comprise block copolymers that include one or more blocks of poly(ethylene glycol) and poly(lactic acid).
  • the invention further provides a method of killing cancer cells by contacting the cells with a composition that includes an effective amount of 17- AAG-containing micelles.
  • the contacting can be in vivo or in vitro.
  • the 17- AAG containing micelles can also be used to prevent, slow, or inhibit the growth of a cancer tumor by contacting, or administering to, the tumor a composition that includes an effective amount of 17-AAG-containing micelles.
  • the invention further provides a method of preparing 17-AAG-containing micelles, wherein 17- AAG and unimers (block polymer chains not in micellar arrangement) are first dissolved in a water-miscible solvent (for example, dimethylacetamide (DMAc)).
  • a water-miscible solvent for example, dimethylacetamide (DMAc)
  • the solution can then be added to a dialysis apparatus (e.g., a bag or tubing).
  • the dialysis apparatus can then be placed in an aqueous bath, to provide the 17-AAG-containing micelles in the dialysis bag.
  • the micelles can then be separated, for example, by centrifugation to precipitate unincorporated drug. Nano-filtration can be used to provide the isolated 17- AAG-containing micelles.
  • the invention also provides a method of preparing 17- AAG-containing micelles where 17-AAG and PEG-PLA (2000 mw; 50:50) are dissolved in a suitable solvent, such as acetonitrile or DMAc.
  • a suitable solvent such as acetonitrile or DMAc.
  • the blocks of the PEG-PLA can be an approximate 50:50 of 2,000 mw blocks.
  • the solution can be sonicated and then concentrated by solvent removal. Warm water (50-70 0 C, or approximately 60 0 C) can then be added and the mixture can be allowed to cool to ambient temperature (-23 0 C). The mixture can then be separated, for example, by centrifugion to remove sediment (e.g., at about 13,000 rpm for about one minute).
  • the supernatant can be collected and filtered, for example, through a 0.2 ⁇ m PTFE filter, to provide the isolated 17-AAG-containing micelle formulation.
  • the resulting micelle pharmaceutical solution can be stored at low temperatures, e.g., about 4 0 C, for extended periods of time.
  • Figure 1 illustrates characterization of micelles, according to an embodiment of the invention
  • DLS dynamic light scattering
  • Figure 2 illustrates in vitro release of 17- AAG from 0.3-mM PEG-PLA micelles in ddH 2 O at 37 0 C and pH 7.4, according to one embodiment.
  • Micelles were prepared with 11.4% w/w 17- AAG based on PEG-PLA content, as noted in Table 1 ; PEG-PLA drug-loaded micelles (•); drug alone (V); and 17-AAG formulated in CrEL-EtOH-PEG400 ( ⁇ ).
  • Figure 3 illustrates pharmacokinetic profiles in rats, including (a) serum, (b) amount of 17-AAG remaining in the urine, (c) rate of urine excretion of 17- AAG, and (d) total amount of 17- AAG excreted through the urine, according to one embodiment.
  • the asterisk (*) denotes statistically significant differences (p ⁇ 0.05) between the standard formulation of CrEL-EtOH-PEG400 and 17-AAG encapsulated in PEG-PLA micelles.
  • FIGS 5 (a) and 5(b) illustrate 17-AAG micelle preparation procedures according to embodiments of the invention.
  • Figure 6 illustrates a characterization of the drug loading of PEG-PLA micelles encapsulating 17-AAG, determined by UV measurements at 337 nm, according to an embodiment.
  • Figure 7 illustrates particle size distribution in PEG-PLA micelles determined by DLS measurement at 632.8 nm, according to one embodiment; (a) number- weight Gaussian distribution, and (b) volume- weight Gaussian distribution.
  • Figure 8 shows a transmission electron microscopy (TEM) image of PEO- b-PDLA micelles (18,000 x).
  • Figure 9 illustrates a micelle loading and formation procedure, according to one embodiment. After nanofiltration the formulation can be analyzed using HPLC with UV and RI detection modes.
  • TEM transmission electron microscopy
  • Figure 10 illustrates the solubilization of paclitaxel and 17- AAG together in PEG-PLA micelles.
  • Figure 11 illustrates physical stability results for 17-AAG/paclitaxel PEG-
  • Figure 12 illustrates (a) solubility of paclitaxel (PTX) and 17-AAG dual agent micelles (initial); (b) solubility of paclitaxel and 17-AAG dual agent micelles after 24 hours; (c) a comparison of paclitaxel concentration in PTX/17- AAG dual agent micelles at 0 hours and at 24 hours; and (d) a comparison of paclitaxel concentration in paclitaxel-only micelles at 0 hours and at 24 hours.
  • PTX solubility of paclitaxel
  • 17-AAG dual agent micelles after 24 hours
  • a comparison of paclitaxel concentration in PTX/17- AAG dual agent micelles at 0 hours and at 24 hours
  • paclitaxel concentration in paclitaxel-only micelles at 0 hours and at 24 hours.
  • Figure 13 illustrates (a) solubility of etoposide (ETO) and 17-AAG dual agent micelles (initial); (b) solubility of etoposide and 17-AAG dual agent micelles after 24 hours; (c) a comparison of etoposide concentration in ETO/17- AAG dual agent micelles at 0 hours and at 24 hours; and (d) a comparison of etoposide concentration in etoposide-only micelles at 0 hours and at 24 hours.
  • ETO solubility of etoposide
  • 17-AAG dual agent micelles after 24 hours
  • a comparison of etoposide concentration in ETO/17- AAG dual agent micelles at 0 hours and at 24 hours
  • a comparison of etoposide concentration in etoposide-only micelles at 0 hours and at 24 hours.
  • Figure 14 illustrates (a) solubility of docetaxel (DCTX) and 17-AAG dual agent micelles (initial); (b) solubility of docetaxel and 17-AAG dual agent micelles after 24 hours; (c) a comparison of docetaxel concentration in DCTX/17-AAG dual agent micelles at 0 hours and at 24 hours; and (d) a comparison of docetaxel concentration in docetaxel-only micelles at 0 hours and at 24 hours.
  • DCTX docetaxel
  • 17-AAG dual agent micelles initial
  • Figure 15 illustrates (a) the physical stability of paclitaxel (PTX) single agent micelles over time, as analyzed by optical density (OD) changes; and (b) the physical stability of paclitaxel/17-AAG dual agent micelles over time, as analyzed by optical density (OD) changes.
  • PTX paclitaxel
  • OD optical density
  • Geldanamycin is a well-known natural product, obtainable by culturing the producing organism, Streptomyces hygroscopicus var. geldanus NRRL 3602.
  • the compound 17-AAG is made semi-synthetically from geldanamycin, by reaction of geldanamycin with allylamine, as described in U.S. Patent No. 4,261,989 (Sasaki et al.), the disclosure of which is incorporated herein by reference.
  • Suitable water solubility is of particular importance for parenteral administration.
  • the water solubility of 17- AAG is only about 0.1 mg/mL at neutral pH, making it difficult to administer in a safe and effective manner. Attempts have been made to address the solubility issue but each formulation was accompanied by its own drawbacks, such as the use of DMSO, ethanol, or various undesirable surfactants.
  • the compound 17-AAG (17-allylamino-17-demethoxygeldanamycin, or tanespimycin) is a promising heat shock protein 90 inhibitor currently undergoing clinical trials for the treatment of cancer.
  • 17-AAG faces challenging issues due to its poor aqueous solubility.
  • Current 17- AAG compositions require formulation with Cremophor EL (CrEL), DMSO, and/or ethanol. See U.S. Application Publication No. 2005/0256097 (Zhong et al.).
  • Cremophor ® EL is a castor oil derivative, typically prepared by reacting 35 moles of ethylene oxide with each mole of castor oil to provide a polyethoxylated castor oil (CAS number 61791- 12-6).
  • the use of Cremophor ® EL (e.g. KOS-953) or DMSO for parenteral formulations is undesirable from a patient tolerability standpoint due to its potential side effects.
  • Various adverse effects can include acute hypersensitivity reactions, peripheral neurotoxicity, hyperlipidaemia, and/or inhibition of P-glycoprotein.
  • 17-AAG has a high volume of distribution (Vd) and considerable systemic toxicity at low doses (less than 20 mg/kg). Accordingly, improved formulations are needed to safely administer 17- AAG to patients in need of such treatment.
  • the disclosure herein provides a CrEL- free formulation of 17-AAG, prepared using amphiphilic diblock micelles composed of poly(ethylene oxide)- £-poly(D,L-lactic acid) (PEG-PLA).
  • Dynamic light scattering (DLS) revealed PEG-PLA (12:6 kDa) micelles with average diameters of about 257 nm and critical micelle concentration of about 350 nM.
  • the micelles can solubilize significant quantities of certain active agents, for example, about 1.5 mg/mL of 17-AAG.
  • the area under the curve (AUC) of PEG-PLA micelles was 1.3-fold that of the standard formulation.
  • the micelle formulations described herein provide delivery vehicles for 17-AAG that have several advantages over currently known compositions.
  • the 17-AAG micellar formulation showed a 2.7-fold increase in the half-life (tj /2 ) of the drug in serum and 1.3-fold increase in ti /2 in urine.
  • V d volume of distribution
  • PEG-PLA refers to poly(ethylene oxide)-b/oc&-poly(lactic acid).
  • the poly(lactic acid) block can include (D-lactic acid), (L-lactic acid), (D,L-lactic acid), or combinations thereof.
  • Various forms of PEG-PLA are available commercially, such as from Polymer Source, Inc., Montreal, Quebec, or they can be prepared according to methods well known to those of skill in the art.
  • the molecular weight of the poly(ethylene glycol) block can be about 1,000 to about 35,000 g/mol, or any increment of about 500 g/mol within said range.
  • Suitable blocks of the poly(lactic acid) can have molecular weights of about 1,000 to about 15,000 g/mol, or any increment of about 500 g/mol within said range.
  • the PEG block can terminate in an alkyl group, such as a methyl group (e.g., a methoxy ether) or any suitable protecting, capping, or blocking group.
  • alkyl group such as a methyl group (e.g., a methoxy ether) or any suitable protecting, capping, or blocking group.
  • PEG-PLA micelles can be prepared as described in Example 1 , as well as schematically illustrated in Figure 5(a).
  • Figure 5(b) provides one specific procedure for preparing a 17-AAG micelle formulation. This procedure is merely illustrative for one embodiment and the procedure can be varied according to the desired scale of preparation, as would be readily recognized by one skilled in the art.
  • One advantage of the procedure illustrated in Figure 5 is that it does not require dialysis of a micelle solution.
  • the micelles of this disclosure can be prepared using PEG-PLA polymers of a variety of block sizes (e.g., a block size within a range described above) and in a variety of ratios (e.g., PEG:PLA of about 1 :10 to about 10:1, or any integer ratio within said range).
  • molecular weights (M n ) of the PEG-PLA polymers can include, but are not limited to, 2K-2K, 3K-5K, 5K-3K, 5K-6K, 6K- 5K, 6K-6K, 8K-4K, 4K-8K, 12K-3K, 3K-121C, 12K-6K, and/or 6K-12K.
  • the drug-to-polymer ratio can also be about 1 :20 to about 10: 1, or any integer ratio within said range.
  • suitable drug-polymer ratios include, but are not limited to, about 1:2.5; about 1:5; about 1:7.5; and/or about 1:10.
  • One suitable polymer is a PEG-PLA that includes blocks of about 1-3 kDa (e.g., about 2K Daltons) at an approximate 50:50 ratio.
  • Use of this procedure resulted in unexpectedly high levels of drug loading in the micelles. For example, when the procedure of Figure 5 was employed, drug loading of about 5 mg/mL was achieved (about 9 mM; about 17 wt%) (see Figure 6).
  • a micelle-encapsulated active agent formulation can also be prepared by a dialysis method.
  • the agent and the PEG-PLA can be dissolved in a suitable water miscible organic solvent, such as dimethyl acetamide (DMAc).
  • DMAc dimethyl acetamide
  • the solution is then transferred to a dialysis bag.
  • the dialysis medium contains an aqueous solution, such as 0.9% saline.
  • the dialysis bag can be, for example, a 3500 MWCO tubing (SpectraPor ® ) dialysis bag.
  • micelles form, incorporating the active agent.
  • the micelle-encapsulated drugs can then be isolated. For example, unincorporated drug can be precipitated by centrifugation.
  • Nano filtration of the resulting supernatant provides the isolated micelle-encapsulated active agent formulation.
  • the micelles can be measured and analyzed, for example, using an HPLC equipped with UV and RI detection modes (see the techniques described by Yasugi et al., J. Control. Release, 1999, 62, 99-100). Preparatory methods can also include the use of oil-in- water emulsions, solution casting, and/or freeze-drying (lyophylization). See Gaucher et al., J. Controlled Release, 109 (2005) 169-188.
  • the micelle-drug composition can be stored for extended periods of time under refrigeration, preferably at a temperature between about -20 0 C and about 4 0 C.
  • Use of brown glass vials or other suitable containers to protect the micelle-drug composition from light can extend their effective lifetime.
  • the micelle-drug compositions can be freeze-dried into a solid formulation, which can be reconstituted with an aqueous vehicle prior to drug administration.
  • a larger amount of the drug can be dissolved in a given amount of fluid, such as a pharmaceutical carrier, or body fluid, such as blood or interstitial fluids, than can be dissolved without use of the micelles.
  • a pharmaceutical carrier that dissolves the micelles such that the micelles can pass through a filter are considered to be dissolved in a pharmaceutical "solution", to provide a formulation according to an embodiment of the invention.
  • the micelles can solubilize up to about 15 mg/mL of 17-AAG, or up to about 20 mg/mL of 17-AAG. In some embodiments, the micelles can solubilize about 3 mg/mL, about 5 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL, or about 25 mg/mL of an active agent. In some embodiments, the formulation can have concentrations of about 0.5 to about 5 mg/mL of 17-AAG, about 0.75 to about 3 mg/mL of 17-AAG, about 1 to about 2 mg/mL of 17-AAG, or about 1.5 mg/mL, with respect to the volume of micelles or preferably, the volume of the aqueous carrier.
  • 17-AAG encapsulated micelles are formulated in a mixture that includes an aqueous carrier, such as saline or dextrose, and the like.
  • a suitable carrier can be 0.9% NaCl solution, or a 5% aqueous saccharide solution, such as a dextrose or glucose solution. See, Remington: The Science and Practice of Pharmacy, D.B. Troy, Ed., Lippincott Williams & Wilkins (21 st Ed., 2005) at pages 803-849.
  • sterile aqueous solutions of water-soluble salts can be employed.
  • Additional or alternative carriers may include sesame or peanut oil, as well as aqueous propylene glycol.
  • Aqueous solutions may be suitably buffered, if necessary, and the liquid diluent can first be rendered isotonic with sufficient saline or glucose.
  • These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intraperitoneal, and intratumoral (IT) injection.
  • Intratumoral injection can be especially helpful for certain types of therapy, such as the treatment of cancer, including prostate cancer.
  • Appropriate sterile aqueous media can be purchased (e.g., Sigma-Aldrich Corporation, St. Louis, MO) or can be prepared by standard techniques well known to those skilled in the art.
  • the compositions are completely free of additives such as one or more of ethanol, dimethyl sulfoxide, or other organic solvents, phospholipids, castor oil, and castor oil derivatives, hi other embodiments, the composition is substantially free of such components.
  • substantially free means that the composition contains less than about 2.5 wt.%, less than about 2 wt.%, less than about 1.5 wt.%, less than about 1 wt.%, less than about 0.5 wt.%, or less than about 0.25 wt.%.
  • certain additives can increase the stability of the micelles.
  • a surfactant can be included in the micelle (e.g., in about 0.25 wt.% to about 2.5 wt.%).
  • a suitable surfactant can be a negatively charged phospholipid, such as polyethylene glycol conjugated distearoyl phosphatidyl- ethanolamine (PEG-DSPE).
  • the micelles can be formulated into a pharmaceutical solution and administered to a patient.
  • the pharmaceutical solution formulation can allow for delivery of a requisite amount of 17- AAG to the body within an acceptable time, for example, about 10 minutes, to about 3 hours, typically about 1 to about 2 hours, for example, about 90 minutes.
  • the administration can be parenteral, for example, by infusion, injection, or rv, and the patient can be a mammal, for example, a human.
  • the micelles can circulate intact, dissociate into individual polymer chains, release active agents (either by diffusion or micelle dissociation), distribute into tissue (e.g. tumors), and/or undergo renal clearance.
  • the schedule of these events cannot be predicted with specificity, and these events significantly influence the anti-tumor activity of the active agents, such as 17- AAG.
  • the drug-loaded micelles can extravasate into tumor interstitial.
  • the active agent-containing micelles can hydrolyze to release the 17-AAG, which can then release the active agent from the micelle.
  • the active agent can then diffuse into tumor cells.
  • Another aspect of the invention includes the micelles crossing leaky vasculature and endocytosing into tumor cells, and inhibiting the tumor cell growth, and/or killing the tumor cells.
  • a disease, disorder, or condition can be treated by administering a pharmaceutical formulation of micelles that contain 17-AAG.
  • Administration of the compositions described herein can result in a reduction in the size and/or the number of cancerous growths in a patient, and/or a reduction in one or more corresponding associated symptoms.
  • the compositions of the invention can produce a pathologically relevant response, such as inhibition of cancer cell proliferation, reduction in the size of a cancer or tumor, prevention of further metastasis, inhibition of tumor angiogenesis, and/or death of cancerous cells.
  • the method of treating such diseases and conditions described below includes administering a therapeutically effective amount of a composition of the invention to a patient.
  • Conditions that can be treated include, but are not limited to, hyperproliferative diseases, including cancers of the head and neck, which include tumors of the head, neck, nasal cavity, paranasal sinuses, nasopharynx, oral cavity, oropharynx, larynx, hypopharynx, salivary glands, and paragangliomas; cancers of the liver and biliary tree, particularly hepatocellular carcinoma; intestinal cancers, particularly colorectal cancer; ovarian cancer; small cell and non-small cell lung cancer; prostate cancer; pancreatic cancer; breast cancer sarcomas, such as fibrosarcoma, malignant fibrous histiocytoma, embryonal rhabdomysocarcoma, leiomysosarcoma, neurofibrosarcoma, osteosarcoma, synovial sarcoma, liposarcoma, and alve
  • Non-cancer conditions that are characterized by cellular hyperproliferation can also be treated using the methods described herein.
  • 17-AAG can be administered according to the methods described herein to treat conditions that are characterized by cellular hyperproliferation.
  • Illustrative examples of such non-cancer conditions, disorders, or diseases include, but are not limited to, atrophic gastritis, inflammatory hemolytic anemia, graft rejection, inflammatory neutropenia, bullous pemphigoid, coeliac disease, demyelinating neuropathies, dermatomyositis, inflammatory bowel disease (ulcerative colitis and/or Crohn's disease), multiple sclerosis, myocarditis, myositis, nasal polyps, chronic sinusitis, pemphigus vulgaris, primary glomerulonephritis, psoriasis, surgical adhesions, stenosis or restenosis, scleritis, scleroderma, eczema (
  • vasculitis e.g., Giant cell arteritis (temporal arteritis, Takayasu's arteritis), polyarteritis nodosa, allergic angiitis and granulomatosis (Churg-Strauss disease), polyangitis overlap syndrome, hypersensitivity vasculitis (Henoch-Schonlein purpura), serum sickness, drug- induced vasculitis, infectious vasculitis, neoplastic vasculitis, vasculitis associated with connective tissue disorders, vasculitis associated with congenital deficiencies of the complement system, Wegener's granulomatosis, Kawasaki's disease, vasculitis of the central nervous system, Buerger's disease and systemic sclerosis; gastrointestinal tract diseases, e.g., pancreatitis, Crohn's disease, ulcerative colitis, ulcerative proctitis, primary sclerosing cholangitis, benign strictures of any cause including ideopathic (e.
  • treat and “treatment” refer to any process, action, application, therapy, or the like, wherein a mammal, including a human being, is subject to medical aid with the object of improving the mammal's condition, directly or indirectly. Treatment typically refers to the administration of an effective amount of a micelle composition as described herein.
  • an effective amount or “therapeutically effective amount” are intended to qualify the amount of a therapeutic agent required to relieve to some extent one or more of the symptoms of a condition, disease or disorder, including, but not limited to: 1) reduction in the number of cancer cells; 2) reduction in tumor size; 3) inhibition of (i.e., slowing to some extent, preferably stopping) cancer cell infiltration into peripheral organs; 3) inhibition of (i.e., slowing to some extent, preferably stopping) tumor metastasis; 4) inhibition, to some extent, of tumor growth; 5) relieving or reducing to some extent one or more of the symptoms associated with the disorder; and/or 6) relieving or reducing the side effects associated with the administration of active agents.
  • inhibition in the context of neoplasia, tumor growth or tumor cell growth, may be assessed by delayed appearance of primary or secondary tumors, slowed development of primary or secondary tumors, decreased occurrence of primary or secondary tumors, slowed or decreased severity of secondary effects of disease, arrested tumor growth and regression of tumors, among others. In the extreme, complete inhibition, can be referred to as prevention or chemoprevention.
  • the inhibition can be about 10%, about 25%, about 50%, about 75%, or about 90% inhibition, with respect to progression that would occur in the absence of treatment.
  • active agents such as 17- AAG and/or an anticancer or cytotoxic agent may be administered in a dose ranging from about 4 mg/m 2 to about 4000 mg/m 2 , depending on the frequency of administration.
  • a dosage regimen for 17- AAG can be about 400-500 mg/m 2 weekly, or about 450 mg/m 2 weekly.
  • Banerji et al. Proc. Am. Soc. Clin. Oncol., 22, 199 (2003, abstract 797).
  • a dose of about 300 mg/m 2 to about 325 mg/m 2 , or about 308 mg/m 2 weekly can be administered to the patient. See Goetz et al., Eur. J.
  • Another dosage regimen includes twice weekly injections, with doses ranging from about 200 mg/m 2 to about 360 mg/m 2 (for example, about 200 mg/m 2 , about 220 mg/m 2 , about 240 mg/m 2 , about 250 mg/m 2 , about 260 mg/m 2 , about 280 mg/m 2 , about 300 mg/m 2 , about 325 mg/m 2 , 340 mg/m 2 , about 350 mg/m 2 , or about 360 mg/m 2 , depending on the severity of the condition and health of the patient).
  • doses ranging from about 200 mg/m 2 to about 360 mg/m 2 (for example, about 200 mg/m 2 , about 220 mg/m 2 , about 240 mg/m 2 , about 250 mg/m 2 , about 260 mg/m 2 , about 280 mg/m 2 , about 300 mg/m 2 , about 325 mg/m 2 , 340 mg/m 2 , about 350 mg/m 2 , or about 360 mg/m
  • a dosage regimen that can be used for combination treatments with another drug, such as docetaxel, can be to administer the two drugs every three weeks, with the dose of 17- AAG of about 500 mg/m 2 to about 700 mg/m 2 , or up to about 650 mg/m 2 at each administration.
  • paclitaxel-containing micelles can be used in a formulation along with 17- AAG- containing micelles (a "simply mixed" micelle formulation, wherein each micelle contains a different active agent).
  • the invention provides a composition that includes both 17-AAG and a second active agent, wherein both the 17-AAG and the second active are solubilized in an aqueous solution by encapsulation within PEG-PLA micelles.
  • the composition can include both paclitaxel and 17-AAG in the micelles of a simply mixed micelle formulation.
  • Formulations can also be prepared where both 17-AAG and a second active agent are dissolved in the micelle preparation procedure, forming individual micelles that contain both 17-AAG and the second active agent, thus providing "physically mixed" micelles, wherein one or more of the micelle contains two different active agent.
  • 17-AAG can be administered (e.g., in micelles as described herein) in combination with other active agents (e.g., anti-cancer or cytotoxic agents), including alkylating agents, angiogenesis inhibitors, antimetabolites, DNA cleavers, DNA crosslinkers, DNA intercalators, DNA minor groove binders, heat shock protein 90 (Hsp 90) inhibitors, histone deacetylase inhibitors, microtubule stabilizers, nucleoside (purine or pyrimidine) analogs, proteasome inhibitors, topoisomerase (I or II) inhibitors, tyrosine kinase inhibitors.
  • active agents e.g., anti-cancer or cytotoxic agents
  • active agents e.g., anti-cancer or cytotoxic agents
  • alkylating agents e.g., angiogenesis inhibitors, antimetabolites, DNA cleavers, DNA crosslinkers, DNA intercalators, DNA minor groove binders, heat shock protein 90 (Hsp
  • Specific active agents include ⁇ -lapachone, 17-DMAG, bicalutamide, bleomycin, bortezomib, busulfan, calicheamycin, callistatin A, camptothecin, capecitabine, carzelesin, CC-1065, cisplatin, clanfen ⁇ r, cryptophycins, cyclosporine A, daunorubicin, diazepam, discodermolide, docetaxel, doxorubicin, duocarmycin, dynemycin A, epothilones, etoposide, floxuridine, fludarabine, fluorouracil, gefitinib, geldanamycin, gemcitabine, hydroxyurea, imatinib, interferons, interleukins, itraconazole, irinotecan, leptomycin B, methotrexate, mitomycin C, oxaliplatin, paclitaxel, spong
  • the co-administered anti-cancer or cytotoxic agent can also be a protein kinase inhibitor.
  • protein kinase inhibitors include rapamycin; quinazolines, particularly 4-anilinoquinazolines such as Iressa (AstraZeneca; N-(3-chloro-4-fluorophenyl)-7-rnethoxy-6-[3-(4- morpholinyl)propoxy]-4-quinazolinamine) or Tarceva (Roche/Genentech; N-(3- ethynylphenyl)-6,7-bis(2-m- ethoxyethoxy)-4-quinazolinamine monohydrochloride); phenylamino-pyrimidines such as Gleevec (Novartis; 4-[(4- methyl-l-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2- pyrimidinyl
  • the combination of 17- AAG and certain other active agents has synergistic anticancer activity.
  • synergy can be observed when 17- AAG is administered in combination with paclitaxel, docetaxel, etoposide, as well as several other agents listed above.
  • these two agents can be combined in the same micelles (physically mixed formulations), or micelles individually incorporating paclitaxel and 17- AAG in separate micelles can be combined into one treatment formulation (a simply mixed formulation) for administration to a patient.
  • the 17-AAG micelles can be administered before, concurrently, or after administration of a drug other than 17- AAG.
  • the drug other than 17-AAG can be administered in by any effective means, including administration by micelle encapsulation as described herein.
  • the 17-AAG can sensitize the patient so that lower amounts of the other drug are necessary for effective treatment.
  • the dual-agent micelles could be prepared such that the drug loading was within about 20% of the maximum loading that was obtainable for single-agent micelles.
  • PEG-PLA micelles that contain both active agents in their cores are more stable with respect to the loss of one of the actives. Accordingly, it was discovered that in micelles containing two active agents, the actives can interact in such a manner as to increase the stability of the micelle, with respect to release of the actives.
  • micelles that contain 17- AAG and a second active agent, such as paclitaxel, docetaxel, or etoposide have been found to be more stable than micelles that incorporate only one of the active agents.
  • 17-AAG a chemotherapeutic agent
  • paclitaxel e.g., paclitaxel, docetaxel, or etoposide, among others
  • a chemotherapeutic agent e.g., paclitaxel, docetaxel, or etoposide, among others
  • DMSO and Cremophor ® EL a four component cocktail.
  • the components of such formulations have been found cause significant adverse side-effects in some patients. Also, the two drugs cannot be mixed and infused together, and any drug synergy is achieved by concurrent drug administration (Solit et al., Cancer Res., 2003;63:2139-2144).
  • paclitaxel/PEG-PLA can be safely administered to patients.
  • Genexol-PM is currently in phase II clinical trials.
  • 17-AAG can be co-loaded into PEG-PLA micelles without requiring a significant increase in the number of the micelles.
  • Such formulations can also avoid the use of organic solvents or other surfactants.
  • amphophilic block copolymer (ABC) micelle systems described herein.
  • Drug synergy can be achieved by use of the micelles, which can reduce the toxicity of a treatment regimen due to drug encapsulation within the micelle delivery vehicles.
  • Combinations of active agents can be used in the micelles. Simply mixed and physically mixed formulations allow for the administration of two different active agents with one administration, e.g., an IV infusion. Certain useful combinations and techniques are described in U.S. Patent No. 7,221,562 (Rosen et al.).
  • Other amphiphilic copolymers that may be used in embodiments of the invention include those described in U.S. Patent No. 4,745,160 (Churchill et al.).
  • the micelle compositions disclosed herein provide for improved formulation that have unexpectedly high loading capacity for 17- AAG and can be used to prepare controlled release formulations. It was also discovered that the drug loading dual-active micelles can approach, or be equal to, the drug loading capacity of single agent micelles. Additionally, interaction between the actives in the dual-active micelles can increase the stability of such micelles. For example, 17-AAG can act as a stabilizer for dual agent micelle formulations, with respect to both simply mixed formulations and also physically mixed formulations.
  • H-13 1.60-1.85 (br m, 6H, H-13, H-14, 8-Me), 2.05 (s, 3H, 2-Me), 2.46 (br m, 2H, H-15), 2.83- 2.90 (br m, 3H, H-10), 3.27 (s, 3H, OMe), 3.36 (s, 3H, OMe), 3.40 (t, IH, H-12), 3.58-3.68 (br m, 2H, H-I l.
  • 17-AAG was formulated by dissolving it with PEG-PLA (12:6 kDa) (Polymer Source, Montreal, Canada) in dimethylacetamide (DMAc) and dialyzing against H 2 O, following procedures by Kataoka and coworkers (J. Control. Release 62(1- 2) (1999) 89-100).
  • PEG-PLA 12:6 kDa
  • DMAc dimethylacetamide
  • 5 mg of 17- AAG and 45 mg of PEG-PLA (10:90 w/w) were dissolved in 10 mL DMAc.
  • the resulting solution was dialyzed against H 2 O in 3500 MWCO tubing (SpectraPor). Resulting micelles were centrifuged at 5000 g's for 10 min to precipitate unincorporated drug.
  • Quantitative drug loading in micelles was determined by monitoring the area under the curve (AUC) for 17-AAG (based on a 17- AAG calibration curve) through reverse-phase HPLC (Shodex C18 column, 65-82.5 : 35-17.5 MeOH to 55% MeOH+0.2% formic acid gradient, 40 °C, 332 nm detection).
  • Effective diameters of PEG-PLA micelles, with and without drugs, were measured using a Brookhaven dynamic light scattering apparatus (100 mW, 532 nm laser) with Gaussian intensity fitting.
  • CMC critical micelle concentration
  • PEO-b-PDLLA micelles were prepared as described above in serial dilutions and incubated with 0.6 ⁇ M pyrene for 1 hour at 80 0 C, allowed to sit in the dark for 15 hours at RT, and the fluorescence emission of pyrene was measured at 390 nm (RF-5301 PC spectrofluorophotometer, Shimadzu). Pyrene undergoes well-known photophysical changes in response to its microenvironment polarity (Colloids Surf., A Physiochem. Eng. Asp. 118 (1996) 1-39).
  • ABSC amphiphilic block copolymer
  • PEG-PLA poly(ethylene oxide)- £/oc£-poly(D,L-lactide)
  • Peristaltic pumps under computer control separately injected 50 g/L solutions of dibasic and monobasic phosphate to maintain pH at 7.4 ⁇ 0.1 (apparatus built in-house).
  • dialysis cassette volumes were made up with ddH 2 O to 2 mL, if necessary, and 100 ⁇ L aliquots were withdrawn. This was mixed with 100 ⁇ L MeOH and 40 ⁇ L of the mixture was analyzed by reverse-phase HPLC (Shodex Cl 8 column; 65- 82.5 : 35-17.5 of A to B where A: MeOH and B: 55% MeOH+0.2% formic acid; 40 °C; 332 nm detection).
  • PC-3 human prostate cells (ATCC CRL- 1435) were grown in RPMI 1640 (Hyclone, Logan, UT) and MCF-7 human breast cancer cells (ATCC HTB-22) were cultured in DMEM (Hyclone), both supplemented with 10% Fetal Bovine Serum (Hyclone), 100 ⁇ g/mL penicillin- streptomycin (Cambrex Biosciences, Baltimore, MD), and 2 mM L-glutamine (Cambrex Biosciences).
  • Cell lines were plated in 96-well plates at an initial density of 3000 cells per well in 90 ⁇ L of appropriate media, and maintained at 37 0 C under a 5% CO 2 atmosphere.
  • the 17-AAG was analyzed at 332 nm with an internal standard at 305 nm on a Genesis 3 ⁇ m Cl 8 33mm x 4.6mm column at 1 mL/min in A: 50 mM acetic acid + 10 mM TEA and B: MeOH + 10 rtiM TEA (0-3 min 40% B, 3.01-11 min 80% B, 11.01-18 min 40%B).
  • Pharmacokinetic parameters were calculated using WinNonlin ® software (ver. 5.01) and non-compartmental modeling. All animal studies were conducted in accordance with "Principles of laboratory animal care" (NIH publication No. 85-23, revised 1985).
  • Tissue samples were blotted with paper towels, washed in ice-cold saline, bottled to remove excess fluid, weighed and rapidly frozen in liquid nitrogen, pulverized to a fine powder with a mortar and pestle under liquid nitrogen, and stored at -70 0 C until assessed for drug concentrations by HPLC analysis.
  • NCSS NCSS Statistical and Power Analysis software
  • the reported values of 17-AAG loading in Table 1 represent 10% w/w loading of 17-AAG based on PEG-PLA.
  • Drug incorporation into micelles was verified using aqueous GPC (Shodex SB-806M) by monitoring equivalent retention times in refractive index (micelles) and 17-AAG (UV ⁇ 332).
  • the mole ratio of 17-AAG to PEG-PLA ranged from 0.73 ⁇ 0.09 to 1, with a 17-AAG loading efficiency of 19 ⁇ 3 % w/w.
  • the maximum solubility of 17-AAG in 0.3-mM PE ⁇ 6 0 oo-b-PDLLA 12 ooo was about 1.5 ⁇ 0.2 mg/mL, an improvement of about 150-fold over 17-AAG ( ⁇ 10 ⁇ g/mL according to the National Cancer Institute).
  • Incorporating a 1 :1 ratio of alpha- tocopherol to drug was attempted to improve loading as previously reported for rapamycin (J. Control. Release 110(2) (2006) 370-377), but the resulting micelles were large (>500 nm) and were unstable. The drug precipitated out of solution after 4-5 hours even at 4 0 C.
  • PEG-PLA micelles at nominal body temperature were investigated by dialysis of drug-loaded micelles against 37 0 C water. Due to the low solubility of 17-AAG, the release medium would have saturated quickly without continuous purging of the bath. Therefore, a constraint in the analysis of the release data was the inability to measure the release of 17-AAG directly from PEG-PLA micelles. Once the drug released from PEG-PLA micelles, it had to diffuse across the dialysis membrane, introducing a second diffusion barrier and complicating the analysis. To determine if the dialysis membrane contributed significantly to the overall release rate, cassettes were loaded with free 17-AAG (initially dissolved in minimal amount of MeOH before being diluted with water) and the release kinetics were measured. The PEG-PLA micelles demonstrated release with a half-life of ca. 4 hours ( Figure 2), a 2-fold improvement over that of the drug alone.
  • b Denotes statistically significant differences (p ⁇ 0.05) between 17-AAG alone and 17-AAG in PEG-PLA micelles. This difference in IC 50 values may be due to prolonged release of the drugs from micelles, as well as the eventual equilibrium reached between release and drug partitioning back into the micellar core in a closed system. Micelles alone showed no apparent toxicity in the cell lines investigated (IC 50 > 10,000 nM).
  • PEG-PLA (12:6 kDa) micelle formulation compared to the standard formulation with CrEL-EtOH-PEG400 are illustrated in Figure 3.
  • the micellar formulation demonstrated increased serum AUC 24-hrs after injection (Figure 3a) as observed by an effective quantification of 17-AAG from the micellar formulation up to 24 hours compared to the control formulation (effective quantification only up to 12 hours before going below the limit of quantification).
  • the micellar formulation also exhibited increased presence of 17-AAG in the urine after 24 hours (Figure 3b), greater rate of renal clearance over 36 horrs (Figure 3c), and higher levels of drug in the urine 48 hours post-injection (Figure 3d).
  • the serum area under the curve (AUC) of PEG-PLA micelles was 1.3-fold that of the standard formulation.
  • PEG-PLA (12:6 kDa) micelle formulation increased the serum half-life (ti /2 ) of the drug 2.7-fold.
  • An increase (1.7 fold) in the volume of distribution (V d ) with the micelle formulation was observed due to its and prolonged presence in the blood as observed by a lower total clearance (1.3-fold decrease), and higher half-life in serum (2.7-fold increase) and in urine (1.2-fold increase) compared to the control standard vehicle formulation.
  • the renal clearance of the drug increased (4.3 fold) with the micelle formulation as compared to the standard vehicle, which demonstrated a higher (1.5 fold) hepatic clearance (CL hepatic).
  • MRT mean residence time
  • the standard formulation includes CrEL, a harmful surfactant known to cause anaphylaxis in patients.
  • Use of a CrEL formulation requires pretreatment with anti-histamines and steroids (Sydor et al., Proc. Nat. Acad. Sci. USA, 2006; 103(46), 17408-13).
  • the micelle formulations according to the invention would not require such pretreatments.
  • the 17- AAG in PEG-PLA micelles was well tolerated in rats. No acute signs of toxicity were observed throughout the length of the study. Also, no mortality was observed with the nanocarrier formulation compared to a 35% mortality within 24 hours observed with the standard formulation of CrEL-EtOH- PEG400. Thus, the 17-AAG in PEG-PLA micelle nanocarrier formulation can retain the pharmacokinetic disposition of 17-AAG without the need for toxic agents such as EtOH and CrEL. The formulation thus provides a new method for using the promising chemotherapeutic agent 17-AAG in cancer therapy. Biodistribution Studies. Quantifiable amounts of 17-AAG were observed in all assayed tissues ( Figure 4(a)).
  • micellar system provided some differences in the tissue distribution of 17- AAG into the brain, heart, and bladder, while similar concentrations to the standard formulation were found in other tissues analyzed.
  • the tissue to serum ratio (K p ) values in all tissues ( Figure 4(b)) for the micellar formulation was lower than the control.
  • This formulation can solubilize about five mg/mL of 17-AAG in PEG-PLA (2:2 kDa) micelles. Similar work with paclitaxel encapsulation into PEG-PLA micelles has demonstrated that this safer micellar formulation can minimize adverse side effects associated with CrEL following administration of the drug to patients. In addition, the nanoscale dimensions will further benefit tumor specificity of the drug through the EPR effect even in the absence of targeting ligands.
  • Example 3 Polymer Composition and Drug Loading Certain specific micelles according to the invention were prepared as illustrated by the data in Tables 4 and 5. Table 4. Various Drug:Polymer Ratios of Prepared PEG-PLA Micelles
  • paclitaxel and 2mg of 17-AAG were solubilized with 5 mg of PEG-PLA Polymer (2K-2K) for the 1:2.5 drug:polymer ratio micelles (i.e., the ratio was calculated by the mass of one drug, not the sum of drugs).
  • Table 5 illustrates the physical stability of various PEG-PLA micelles encapsulating paclitaxel (PAX), 17-AAG, or the two drugs loaded together.
  • the values in the table indicate the amount of active agent retained by the micelles after 24 hours in distilled water storage at room temperature.
  • Micelles that encapsulate 17-AAG, either alone or in combination with paclitaxel were significantly more stable the paclitaxel single agent micelles. Similar stability results were obtained for micelles containing combinations of 17-AAG/docetaxel or 17-AAG/ etoposide (see Figures 12-15).
  • Figure 10 illustrates the amount of drug formulation that can be dissolved in deionized water (mg/mL).
  • PEG-PLA micelles with block sizes of 5K:6K suitably solubilize either paclitaxel alone or 17-AAG alone at about 5 mg/mL.
  • the paclitaxel micelles however, lose the drug from the micelle core faster than 17-AAG micelles (over a period of 24 hours) by precipitation of paclitaxel from the micelles (e.g., loss of paclitaxel aqueous solubility). It was found that the 17- AAG micelles are significantly more stable and soluble than micelles containing certain other active agents alone (e.g., paclitaxel). Accordingly, 17-AAG can be considered a micelle stabilizer.
  • PEG-PLA micelles that contain both drugs in their cores were shown to be more stable with respect to the loss of the non- 17-AAG active agent (see Figure 11). It was discovered that an interaction between the active agents in the cores of PEG-PLA micelles, for example, 17-AAG and paclitaxel, increased the micelle stability. Polymeric micelles with 5K-6K and 12K-6K PEG-PLA showed added stability at a 1 :7.5 drug:polymer ratio of micelles. The same result was found for micelles that included 17-AAG along with docetaxel or etoposide.
  • PEG-PLA provided lower drug solubilization results at below a 1:5 drug:polymer ratio.
  • Micelles prepared from 5K-3K, 5K-6K, and 12K-6K PEG-PLA provided the highest solubilization at a 1 :7.5 drug:polymer ratio.
  • Etoposide and docetaxel can be co-solubilized with 17-AAG in the same way as paclitaxel and 17-AAG. Also, more etoposide stays in solution over 24 hours in the presence of 17-AAG.
  • Figures 12-15 illustrates optical density changes upon the precipitation of a second active agent (paclitaxel, docetaxel, or etoposide), due to certain amounts of degradation, including drug release from PEG-PLA micelles in water. Micelles containing 17-AAG prevent and/or inhibit this degradation from occurring.
  • a Malvern Zetasizer was used for generating particle size data.
  • Figure 12(c) shows that more than 98% of PAX and more than 97% of the 17-AAG remained in the micelles after 24 hours.
  • Figure 12(d) shows that only 8% of PAX remained in the PAX-alone micelles after 24 hours, indicating that the presence of 17-AAG provides significant stability to the physically mixed micelles.
  • Figures 15(a) and 16(b) further illustrate the additional stability provided to micelles when 17- AAG is incorporated into micelles, in addition to paclitaxel.
  • Measurements of optical density (OD) changes in the drug-micelles were made at 650 nm(RT) using a Varian ® Cary 50 with a dip probe (0 to 1440 minutes; every 15 minutes; acquisition: 0.1 seconds).
  • a 1 :7.5 drug:polymer ratio was used with 5K:6K PEG-PLA micelles.
  • Paclitaxel encapsulated micelles were loaded with 13.4% paclitaxel (wt./wt.) and paclitaxel/17- AAG dual-agent micelles were loaded at 26.8% drug loading (combined; 13.4% each drug).
  • Figure 15(a) shows that significant amounts of paclitaxel were lost from the paclitaxel encapsulated micelles, while paclitaxel/17-AAG micelles were substantially stable over 24 hours ( Figure 15(b)). Further unexpected results were observed when simply mixed micelle compositions were prepared. For example, PEG-PLA micelles were prepared where a sample of micelles encapsulated paclitaxel and a second sample of micelles encapsulated 17-AAG. When the two samples of micelles were combined into a single aqueous formulation, the paclitaxel single agent micelles were more stable against precipitation than a purely paclitaxel single agent formulation of PEG-PLA micelles.
  • the paclitaxel encapsulated micelles in a formulation with 17-AAG micelles had increased stability compared to a formulation made of only paclitaxel incorporated PEG-PLA micelles, hi some embodiments, equilibration of active agents may take place between micelles in a formulation.
  • Other single agent micelles can be similarly stabilized by forming simply mixed formulations with 17-AAG encapsulated PEG-PLA micelles.

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Abstract

L'invention se rapporte à des molécules de 17-AAG encapsulées dans des particules de type micelles. Les micelles peuvent être composées de polyéthylène glycol-bloc-polyacide lactique (PEG-PLA) sûr. Un avantage significatif du PEG-PLA en tant que véhicule est qu'il est moins toxique que le Cremophor® EL ou que le DMSO. De plus, les micelles de PEG-PLA sont plus faciles à manipuler que le DMSO et elles ne dégagent pas de mauvaises odeurs, problème qui se pose actuellement avec les formulations de 17-AAG lors des essais cliniques. L'invention se rapporte aussi à des procédés pour préparer des agents actifs encapsulés dans des micelles et des méthodes thérapeutiques consistant à utiliser les micelles et leurs formulations correspondantes, par exemple pour inhiber la Hsp 90 et/ou pour traiter le cancer.
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KR20100057007A (ko) 2010-05-28
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WO2009009067A2 (fr) 2009-01-15
US20100203114A1 (en) 2010-08-12

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